3.3 Cryopreservation
of Mussel (Mytilus
galloprovincialis)
Sperm
1. Spawn the mussels, and when a male is located, separate the
individual into a dry beaker, and prevent the mussel from
closing the valves completely so the concentrated sperm can
be collected (see Note 3).
2. Dilute the sperm 1:1 with cryoprotecting agent solution (final
concentration 10% (v/v) dimethyl sulfoxide), and suck the
mixture into 0.25 mL straws.
3. Allow equilibration for 5 min at room temperature (18
C)
while loading the straws.
4. Place the straws into the floating rack in a polystyrene box 5 cm
above the liquid nitrogen level for 8 min (for cryopreservation
at an approximate cooling rate of ~20
C/min).
5. Plunge into liquid nitrogen. Store in liquid nitrogen as long as
needed.
6. Thawing can be performed by immersion of the straws in a
controlled temperature water bath (35
C) until the samples
have melted (6 s).
7. Dilute the CPA with seawater slowly to avoid osmotic shock
(4% FSW addition in each step) until motility is regained.
8. Post-thaw quality assessment: When performing a visual examination of the thawed sperm, cells are slow; there is aggregation
of cells with no movement. With cryopreserved sperm, the
fertilization needs at least 100:1 sperm ratio to yield around
50% fertilization success after a contact time of 15 min. Mussels
are sensitive to polyspermy; therefore, cells should be rinsed
from the excess of sperm before incubation (see Note 4).
3.4 Cryopreservation
of Oyster (Crassostrea
angulata) Sperm
1. Open the oyster using an oyster knife and determine the sex
microscopically.
2. Collect the sperm using the dry method, directly from the
gonad using a scalpel to make small cuts in the gonad (see
Fig. 4). A micropipette is used to collect sperm into an
Eppendorf tube.
3. Filter the sperm through 100 μm and 20 μm sieves, and dilute
1:10 in artificial seawater. Assess cell concentration and total
motility. According to prior concentration, sperm is diluted in
order to have a final concentration between 1 and 2 Â 10
9
spermatozoa/mL. Only samples with motility higher than 40%
and final concentration between 1 and 2 Â 10
9 spermatozoa/
mL should be cryopreserved.
4. For cryopreservation, dilute sperm in cryoprotecting solution
1:1 (v:v). During the 10-min equilibration time, place the
obtained suspension into 0.5 mL plastic straws (Minitube,
Germany or IMV, France). If the straws are to be used for
420
Estefania Paredes et al.
of Mussel (Mytilus
galloprovincialis)
Sperm
1. Spawn the mussels, and when a male is located, separate the
individual into a dry beaker, and prevent the mussel from
closing the valves completely so the concentrated sperm can
be collected (see Note 3).
2. Dilute the sperm 1:1 with cryoprotecting agent solution (final
concentration 10% (v/v) dimethyl sulfoxide), and suck the
mixture into 0.25 mL straws.
3. Allow equilibration for 5 min at room temperature (18
C)
while loading the straws.
4. Place the straws into the floating rack in a polystyrene box 5 cm
above the liquid nitrogen level for 8 min (for cryopreservation
at an approximate cooling rate of ~20
C/min).
5. Plunge into liquid nitrogen. Store in liquid nitrogen as long as
needed.
6. Thawing can be performed by immersion of the straws in a
controlled temperature water bath (35
C) until the samples
have melted (6 s).
7. Dilute the CPA with seawater slowly to avoid osmotic shock
(4% FSW addition in each step) until motility is regained.
8. Post-thaw quality assessment: When performing a visual examination of the thawed sperm, cells are slow; there is aggregation
of cells with no movement. With cryopreserved sperm, the
fertilization needs at least 100:1 sperm ratio to yield around
50% fertilization success after a contact time of 15 min. Mussels
are sensitive to polyspermy; therefore, cells should be rinsed
from the excess of sperm before incubation (see Note 4).
3.4 Cryopreservation
of Oyster (Crassostrea
angulata) Sperm
1. Open the oyster using an oyster knife and determine the sex
microscopically.
2. Collect the sperm using the dry method, directly from the
gonad using a scalpel to make small cuts in the gonad (see
Fig. 4). A micropipette is used to collect sperm into an
Eppendorf tube.
3. Filter the sperm through 100 μm and 20 μm sieves, and dilute
1:10 in artificial seawater. Assess cell concentration and total
motility. According to prior concentration, sperm is diluted in
order to have a final concentration between 1 and 2 Â 10
9
spermatozoa/mL. Only samples with motility higher than 40%
and final concentration between 1 and 2 Â 10
9 spermatozoa/
mL should be cryopreserved.
4. For cryopreservation, dilute sperm in cryoprotecting solution
1:1 (v:v). During the 10-min equilibration time, place the
obtained suspension into 0.5 mL plastic straws (Minitube,
Germany or IMV, France). If the straws are to be used for
420
Estefania Paredes et al.